temporal modulation of β-catenin signaling by multicellular aggregation kinetics impacts embryonic...

Temporal Modulation of b-Catenin Signalingby Multicellular Aggregation Kinetics Impacts

Embryonic Stem Cell Cardiomyogenesis

Melissa A. Kinney,1,* Carolyn Y. Sargent,1,* and Todd C. McDevitt1,2

Pluripotent stem cell differentiation recapitulates aspects of embryonic development, including the regulation ofmorphogenesis and cell specification via precise spatiotemporal signaling. The assembly and reorganization ofcadherins within multicellular aggregates may similarly influence b-catenin signaling dynamics and the asso-ciated cardiomyogenic differentiation of pluripotent embryonic stem cells (ESCs). In this study, dynamicchanges in b-catenin expression and transcriptional activity were analyzed in response to altered cell adhesionkinetics during embryoid body (EB) formation and differentiation. Modulation of intercellular adhesion kineticsby rotary orbital mixing conditions led to temporal modulation of T-cell factor/lymphoid enhancer-bindingfactor activity, as well as changes in the spatial localization and phosphorylation state of b-catenin expression.Slower rotary speeds, which promoted accelerated ESC aggregation, resulted in the early accumulation ofnuclear dephosphorylated b-catenin, which was followed by a decrease in b-catenin transcriptional activityand an increase in the gene expression of Wnt inhibitors such as Dkk-1. In addition, EBs that exhibited increasedb-catenin transcriptional activity at early stages of differentiation subsequently demonstrated increased ex-pression of genes related to cardiomyogenic phenotypes, and inhibition of the Wnt pathway during the initial 4days of differentiation significantly decreased cardiomyogenic gene expression. Together, the results of thisstudy indicate that the expression and transcriptional activity of b-catenin are temporally regulated by multi-cellular aggregation kinetics of pluripotent ESCs and influence mesoderm and cardiomyocyte differentiation.

Introduction

Pluripotent embryonic stem cells (ESCs) are a prom-ising cell source for therapies that are aimed at treating

degenerative and chronic diseases in which native tissues aredamaged beyond their endogenous capacity for repair. Elu-cidating the mechanisms regulating ESC fate decisions willsignificantly aid in the development of directed differentia-tion approaches, in order to produce large quantities of dif-ferentiated cells for regenerative therapies. Differentiation ofESCs is commonly initiated by the spontaneous aggregationof cells via E-cadherin, a Ca2 + -dependent homophilic adhe-sion molecule [1]. The resulting multicellular aggregates,termed ‘‘embryoid bodies’’ (EBs), recapitulate morphogenicevents that are similar to those of preimplantation stage em-bryos, including differentiation into cell phenotypes com-prising the three germ lineages (ectoderm, endoderm, andmesoderm) [2,3]. Although EBs often lack the spatial organi-zation to directly mimic the regulation of tightly controlledspatiotemporal signaling exhibited in vivo, ESCs maintain the

capacity to respond to similar molecular cues, thus motivatingthe analysis of developmentally relevant signaling pathwaysin the context of stem cell differentiation [4].

Transcriptional activation induced by the b-catenin sig-naling pathway controls cell fate decisions during earlyembryonic development, aiding in the regulation of embry-onic patterning and axis formation [5], primitive streak for-mation [6], and mesoderm differentiation [7]. Similarly, inESCs, b-catenin signaling is required for the maintenance ofpluripotency [8,9], and mesoderm differentiation [10], as wellas self-organization and axis formation within EBs [11]. Twomain pools of b-catenin are present within cells [12]: (i) b-catenin sequestered at the cell membrane within adherensjunctions, specifically as a mediator between cadherins andthe actin cytoskeleton [13–15], and (ii) stabilized cytoplasmicb-catenin, which mediates transcription on destabilizationand translocation to the nucleus [16,17]. Canonical Wnt sig-naling is a well-studied pathway that is involved in the ini-tiation of b-catenin-regulated transcription [18]. In theabsence of Wnt, cytoplasmic b-catenin is phosphorylated by

1The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology/Emory University, Atlanta, Georgia.2The Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia.*These authors contributed equally to this work.

STEM CELLS AND DEVELOPMENT

Volume 22, Number 19, 2013

� Mary Ann Liebert, Inc.

DOI: 10.1089/scd.2013.0007

2665

the glycogen synthase kinase 3b (GSK3b)/adenomatouspoliposis coli (APC)/Axin complex and is targeted for ubi-quitination [5,19–21]. On Wnt binding to members of theseven-transmembrane Frizzled receptor family, the Frizzledreceptor heterodimerizes with low-density lipoprotein re-ceptor-related protein 5 and/or 6 (LRP5/6) [22], which, inturn, causes the disruption of the GSK3b/APC/Axin com-plex [23], resulting in the accumulation of stabilized, cyto-plasmic b-catenin [24,25]. The cytoplasmic stabilizationof dephosphorylated b-catenin permits the translocation ofb-catenin to the nucleus, where it acts as a transcriptionalco-activator with T-cell factor/lymphoid enhancer-bindingfactor (TCF/LEF) transcription factors [17] to regulate thetranscription of target genes involved in a wide range ofcellular processes, including cardiac specification and mor-phogenesis during embryonic development [26,27] and ESCdifferentiation [28–30]. Thus, b-catenin is a central compo-nent in cell–cell interactions and gene transcription, both ofwhich are key factors regulating embryonic and ESC mor-phogenesis.

Although the presence of Wnt ligands and the destabili-zation of the GSK3b/APC/Axin complex are required topermit the transcriptional activity of b-catenin, the temporalonset and duration of signaling may also be modulated bythe presence of E-cadherin adhesions between cells, whichsequester b-catenin at the membrane [31,32]. Studies haveillustrated the potential for cross-talk between b-catenin as-sociated with E-cadherin and its availability to participate insignaling and transcriptional activities [33]; therefore, theintercellular adhesion events mitigating initial EB formationmay also regulate downstream b-catenin-regulated tran-scription, thus potentially modulating the cardiomyogenicpotential of the ESC population.

The formation and maintenance of EBs in rotary orbitalsuspension culture produces increased yields of homoge-neous EB populations [34–37] and is a relatively facile tech-nique to modulate ESC aggregation kinetics by simplyvarying the orbital mixing speed [36]. Interestingly, previousstudies demonstrated that the extent of cardiomyogenicdifferentiation in rotary cultures was modulated by the or-bital speed, with the conditions promoting accelerated EBformation kinetics also exhibiting increased cardiomyocytedifferentiation [36]. Therefore, the control of ESC aggregationafforded by the rotary orbital suspension culture platformenables a systematic study of aggregation-induced modula-tion of b-catenin signaling kinetics and the associated regu-lation of cardiomyocyte differentiation.

The objective of the present study was to investigate thedynamics of b-catenin transcriptional activity in response tomodulation of ESC aggregation kinetics during EB forma-tion. b-catenin protein expression and localization were as-sessed via immunostaining for total and dephosphorylatedb-catenin isoforms and immunoblotting of cytoplasmic andnuclear protein fractions, in conjunction with analysis oftranscriptional activity using stably transduced luciferaseTCF/LEF reporter mouse ESCs. In addition, gene expressionof downstream targets of b-catenin signaling and cardio-myocyte differentiation were analyzed to determine the re-lationship between b-catenin protein expression, localization,Wnt/b-catenin pathway signaling dynamics, and differenti-ated phenotypes. The findings of this work demonstrate thatthe dynamics of ESC aggregation during multicellular assem-

bly via cadherins modulates the b-catenin signaling pathwayand alters cardiomyogenic gene transcription in ESCs.

Materials and Methods

ESC culture

Murine ESCs (D3 cell line) were cultured on 0.1% gelatin-coated tissue culture-treated plates (Corning). Culture mediaconsisted of Dulbecco’s modified Eagle’s medium (DMEM)supplemented with 15% fetal bovine serum (Hyclone),100 U/mL penicillin, 100 mg/mL streptomycin and 0.25 mg/mL amphotericin (Mediatech), 2 mM L-glutamine (Media-tech), 1 · MEM nonessential amino acid solution (Media-tech), 0.1 mM 2-mercaptoethanol (Fisher), and 103 U/mL ofleukemia inhibitory factor (LIF; ESGRO, Chemicon). Cul-tures were re-fed with fresh media every other day, andpassaged at approximately 70% confluence.

EB formation and culture

EBs were formed, as previously described, by inoculatinga single suspension of ESCs at 2 · 105 cells/mL into 100 mmbacteriological grade polystyrene Petri dishes with 10 mLESC media without LIF [34]. Dishes were incubated at 37�Cwith 5% CO2 either statically or placed on rotary orbitalshakers (Lab-Line Lab Rotator, Barnstead International) at25, 40, or 55 rpm to impart hydrodynamic conditions [36].90% of media was exchanged every 2 days by collecting theEBs via gravity sedimentation and re-suspending the cul-tures in fresh media.

Whole-mount EB immunostaining

EBs were collected by sedimentation at days 1, 2, 4, and 7of differentiation, rinsed 3 · with phosphate-buffered saline(PBS), and formalin (4% formaldehyde) fixed at room tem-perature for 45 min with rotation. EBs were then rinsed 3 ·(5 min with rotation) in EB wash/block buffer (2% BSA/0.1%Tween-20 in PBS) and permeabilized in 0.05% Triton X-100and 2% BSA solution for 1 h at 4�C with rotation. EBs wereblocked in wash/block buffer for 2 h at 4�C with rotation.After permeabilization and blocking, EBs were incubated withpolyclonal rabbit anti-b-catenin (Millipore; 1:200) and mono-clonal mouse anti-active b-catenin (Millipore; 1:50) specific todephosphorylated Ser-33 and Thr-41 [19] (anti-abC, Millipore;1:50) or with monoclonal rat anti-E-cadherin (Sigma, 1:200) at4�C overnight with rotation, rinsed in wash buffer, and in-cubated with secondary antibodies (Alex Fluor 488 anti-rabbitand Alexa Fluor 546 anti-mouse, Invitrogen; 1:200) for 4 h at4�C with rotation. EBs were rinsed, counterstained withHoechst (1:100), and imaged with a multiphoton laser scan-ning confocal microscope (Zeiss LSM 510 NLO).

Quantification of immunostaining

The relative intensity of expression of E-cadherin, b-cate-nin, and abC was quantified with CellProfiler image analysissoftware [38] using a custom-written script. Briefly, theoutlines of individual cells were determined based on pri-mary identification of nuclei followed by expansion of thebounded region to determine cell boundaries. The intensityof staining for E-cadherin, b-catenin, and abC was then de-termined on a per cell basis, with approximately 50–100 cells

2666 KINNEY, SARGENT, AND MCDEVITT

per EB. The total expression levels for each experimentalcondition were calculated from the average of at least threeindividual representative EBs.

Luciferase transduction

Lentiviral transduction of mESCs with a luciferase TCF/LEF reporter construct (Cignal� TCF/LEF luciferase re-porter; SA Biosciences) was performed to generate stablytransduced puromycin resistant clones that expressed lucif-erase in response to b-catenin signaling. ESCs were plated at50,000 cells/well on 0.1% gelatin-coated tissue culture trea-ted six-well dishes (Corning) and cultured for 24 h before theintroduction of Cignal Lenti vectors. Stable transduction ofD3 ESCs was accomplished by incubating cells with Cignal�Lenti TCF/LEF vectors at Multiplicity of Infection (MOI)levels (3, 10, or 25) for 24 h in the presence of 1 mg/mLpolybrene (Sigma). ESCs were incubated with lentivirusparticles for 24 h, and after an additional 72 h, transducedcells were selected using 3 mg/mL of puromycin (Sigma);concentration was determined from a kill curve using 0.5–3.0 mg/mL. After transduction and selection, individualcolonies were chosen using cloning rings and 0.05% trypsin-EDTA, and plated onto 0.1% gelatin-coated tissue culture-treated 100 mm dishes. The selected clones were maintainedin ESC media supplemented with 3mg/mL puromycin for anadditional 2 weeks and assessed for luciferase expression viaanti-luciferase immunostaining, luciferase activity (with andwithout LiCl treatment), and alkaline phosphatase activity.

ESC immunostaining

Transduced clones and naıve D3s were cultured on 0.1%gelatin-adsorbed tissue culture-treated polystyrene six-welldishes. At *70% confluence, cells were rinsed 3 · with PBS,formalin fixed in the wells for 10 min, and rinsed again withPBS. At *70% confluence, ESCs were fixed with formalin(4% formaldehyde), rinsed with PBS, permeabilized, andblocked with 0.05% Triton X-100/2% BSA/PBS solution for1 h at room temperature. ESCs were then incubated over-night at 4�C with polyclonal rabbit anti-b-catenin (Millipore;1:200), monoclonal rat anti-E-cadherin (Sigma; 1:200) [19],monoclonal mouse anti-luciferase (Santa Cruz Biotech; 1:50),or polyclonal goat anti Oct-4 (Santa Cruz Biotech; 1:100).Cells were rinsed with PBS 3 · , and then incubated withAlexa Fluor-conjugated secondary antibodies (Invitrogen;1:200) for 2 h at room, counterstained with Hoechst (10mg/mL), mounted, and cover slipped. To assess alkaline phos-phatase activity, ESCs were stained using the Vector Red al-kaline Phosphatase Substrate kit (Vector Laboratories). Briefly,cells were incubated in Vector Red substrate in 100 mM Tris-HCl buffer to allow color development for 30 min at roomtemperature while protected from light. After color develop-ment, cells were rinsed with Tris-HCl buffer and water,mounted, and cover slipped. Images were acquired with aNikon TE 2000 inverted microscope (Nikon, Inc.) with aSpotFlex camera (Diagnostic Instruments) or an EVOS fl in-verted microscope (Advanced Microscopy Group).

Luciferase activity quantification

ESCs were cultured with or without the addition of LiCl(25 mM) for 24 h to disrupt the GSK3b/APC/Axin complex

and inhibit the phosphorylation of b-catenin, ultimately re-sulting in increased TCF/LEF activity within transducedcells [39]. Luciferase activity was quantified using the Luci-ferase Assay System (Promega), according to manufacturer’sinstructions. Briefly, ESCs or EBs were rinsed 3 · in PBS,lysed with rotation at 4�C for 10 min, vortexed for 5 s, andcentrifuged at 10,000 rcf for 5 min. The supernatant wascollected and transferred to a prechilled microcentrifugetube. Twenty microliters of the cell lysate was added to100mL of Luciferase Assay Reagent, and luminescence wasdetected using a Femtomaster FB12 luminometer (ZyluxCorporation). Relative light units were normalized to mg ofDNA per sample as determined by Quant-It PicoGreen assay(Invitrogen).

Protein fractionation

EBs were collected at days 2 and 4 of differentiation forwestern blot analysis. The NE-PER cell fractionation kit(Pierce) was used to separate the cytoplasmic and nuclearfractions of undifferentiated cells and differentiating EBs.Briefly, cells and EBs were lysed using CER I reagent sup-plemented with 500 · protease inhibitor cocktail (Calbiochem)and 50 · phosphatase inhibitor cocktail (Calbiochem), fol-lowed by addition of the CER II reagent. After centrifugationat 16,000 rcf, the cytoplasmic supernatant fraction was col-lected. The remaining nuclear pellet was incubated in the NERreagent containing 500 · protease and 50 · phosphatase in-hibitors, and centrifuged at 16,000 rcf for 10 min followed bycollection of the supernatant containing the nuclear fraction.

Western blotting

Sample protein concentration was determined using theBCA Protein Quantification kit (Pierce); equal amounts ofprotein per sample (10mg for cytoplasmic fractions; 35 mg fornuclear fractions) were mixed with loading buffer (0.1 MTris-HCl containing sodium dodecyl sulfate (SDS), glycerol,biomophenol blue, and b-mercaptoethanol); incubated at95�C for 5 min; and loaded in 4%–15% Mini-PROTEAN TGXgels (Bio-Rad). Vertical electrophoresis was performed usingthe Mini-PROTEAN Treta Cell (Bio-Rad) system with SDS/polyacrylamide gel electrophoresis (PAGE) running buffer(Tris base/glycine/SDS solution) at 200V for 30 min. Aprotein ladder (Precision Plus Protein Kaleidoscope, 10–250kDa; Bio-Rad) was also loaded into each gel as a molecularweight reference.

After SDS/PAGE separation, protein was transferred to anitrocellulose membrane (Bio-rad) via semi-dry transfer(Trans-Blot SD; Bio-Rad) at 25V for 20 min. Membranes werethen blocked in near infrared blocking medium (RocklandImmunochemicals) for 1 h and subsequently incubated withprimary antibodies for b-catenin (polyclonal rabbit anti-b-catenin; Millipore; 1:400) and loading controls for cytoplas-mic (polyclonal rabbit anti-GAPDH; Pierce; 1:400) and nu-clear (monoclonal mouse anti-TATA binding protein, TBP;Abcam; 1:2000) fractions overnight at 4�C. Primary antibodyincubation was followed by washes in 0.01% Tween-20/PBSsolution, and membranes were incubated with IR secondaryantibodies (680 anti-mouse and anti-rabbit, LiCor Bios-ciences; 1:2000), followed by washes in PBS/0.1% Tween-20solution. Blots were imaged using the Odyssey Infraredimager (LiCor Biosciences).

MODULATION OF b-CATENIN BY ESC MULTICELLULAR AGGREGATION 2667

Quantitative PCR

Expression of Wnt pathway agonists and antagonists, aswell as cardiogenic genes were assessed via quantitative PCRfrom EBs formed with different aggregation kinetics, as wellas for those supplemented with Wnt inhibitors (5 mM In-hibitor of Wnt Production-4, IWP-4; Stemgent) during thefirst 4 days of differentiation [28]. RNA was extracted fromundifferentiated ESCs and from EBs using the RNeasy Minikit (Qiagen Incorporated). Reverse transcription for com-plementary DNA synthesis was performed from 1mg RNAusing the iScript cDNA synthesis kit (Bio-Rad), and quantita-tive PCR was performed with SYBR green technology on theMyiQ cycler (Bio-Rad). Primer sequences and annealingtemperatures for Wnt-1, Wnt-3a, Dickkopf-1 (Dkk-1), Brachyury T(B-T), mesoderm posterior 1 (Mesp-1), myocyte enhancer factor-2c(Mef-2c), Nkx2.5, a-myosin heavy chain (a-MHC), myosin lightchain-2 ventricle (MLC-2v), and 18S ribosome, are listed in Sup-plementary Table S1; (Supplementary Data are available onlineat www.liebertpub.com/scd). Each primer set was designedwith either Beacon Designer software (Invitrogen) (B-T, Mesp-1,Mef-2c, a-MHC, and MLC-2v) or the Integrated DNA technolo-gies INC design Website (www.idtdna.com) (Wnt11, Wnt3a,Dkk-1, and Nkx2.5) and validated with appropriate cell controls.In order to account for variability in expression of housekeepinggenes, absolute gene expression concentrations were calculatedagainst standard curves and represented per mg of total RNA.

Statistical analysis

Experimental conditions were examined with triplicatesamples for a minimum of two independent experiments, andthe data values presented reflect the mean value – standarderror. One- or two-way analysis of variance was performed todetermine statistical significance (P < 0.05) between experi-mental groups and time points, and where significant, it wasfollowed by post-hoc Tukey analysis to define statistical dif-ferences (P < 0.05) between specific experimental variables.

Results

Spatial localization of E-cadherin and associationwith b-catenin in ESCs

Previous studies have demonstrated that the Wnt signal-ing pathway is an important regulator of pluripotency, andthat nuclear accumulation of b-catenin via treatment using aGSK-3 inhibitor aids in the maintenance of ESC self-renewal[8]. Consistent with published reports, undifferentiated

mESC colonies expressed b-catenin, which was co-localizedwith E-cadherin at the plasma membrane, as well as ex-pressed within the cytoplasm and nucleus (Fig. 1). The spa-tial distribution of both proteins was similar throughout themajority of cells in the colony, with the exception of cellsexhibiting pyknotic nuclei, which demonstrated disruptedpatterns of both b-catenin and E-cadherin. In addition, cellsat the perimeter of colonies expressed decreased b-catenin atthe membrane, which was concurrent with decreases in E-cadherin in regions lacking intercellular connections. Overall,the spatial localization of b-catenin and E-cadherin indicatesthe existence of both molecules during undifferentiated ex-pansion before EB formation, as well as the localization of b-catenin both at the membrane (localized with E-cadherin)and in the cytoplasm and nucleus.

Spatiotemporal expression of E-cadherinin response to EB formation kinetics

On aggregation and continued differentiation of EBs inrotary orbital suspension culture, E-cadherin was localized tothe cell membrane at sites of intercellular connections, andwas relatively homogeneously expressed throughout by mostcells in the EB (Fig. 2). Altering the kinetics of cell–cell ag-gregation via changes in rotary orbital speed [36] suggestedthe modulation of E-cadherin expression during the course ofdifferentiation. Specifically, the delayed aggregation kineticsof EBs formed at 55 rpm led to significantly decreased E-cadherin expression after 1 day (Fig. 2D), compared with25 rpm rotary orbital conditions, which support more rapidEB formation (Fig. 2B). In addition, while the expression of E-cadherin appeared to decrease over 4 days of differentiation at25 rpm (Fig. 2L), cultures maintained at 40 and 55 rpm (Fig.2M, N) exhibited significantly increased expression of E-cad-herin at the cell membrane. Taken together, b-catenin was co-localized with E-cadherin in undifferentiated ESCs, and on EBformation, E-cadherin localization was temporally modulatedin response to altered cell association kinetics.

Spatiotemporal expression of b-catenin in responseto EB formation kinetics

Based on the temporal modulation of E-cadherin and itsestablished association with b-catenin, the spatial patterns oftotal (b-catenin + , red in Fig. 3) and dephosphorylated,transcriptionally active b-catenin (abC + , green in Fig. 3)were assessed within EBs maintained in different cultureconditions during the first 7 days of differentiation. Overall,static EBs exhibited significantly increased expression of

Table 1. Correlation of E-Cadherin and B-Catenin Expression Dynamics During EB Differentiation

Day of differentiation Protein expression Pearson’s r P-value

2 E-cadherin Total B-catenin 0.80 0.20Active B-catenin 0.99 0.01*polyacrylamide gel

electrophoresis4 E-cadherin Total B-catenin 20.97 0.03*

Active B-catenin 0.03 0.977 E-cadherin Total B-catenin 0.67 0.33

Active B-catenin 0.85 0.15

Values in bold denote correlations that are statistically significant. *P < 0.05.


b-catenin at early and late time points, compared with rotaryconditions (Fig. 3A, E). Moreover, EBs maintained in staticconditions exhibited nuclear expression of abC at day 2 ofdifferentiation (Fig. 3A, inset), accompanied by intercellularb-catenin expression at the cell membrane. Conversely, EBsfrom rotary orbital suspension cultures exhibited more ho-mogeneous expression patterns of b-catenin and abC overtime, in which b-catenin was distinctly located either withinthe nucleus or at the cell membrane (Fig. 3B–D, H–J, N–P).After 2 days of differentiation, significantly decreased ex-pression of b-catenin and abC was observed within rotaryEBs (Fig. 3B, C, inset), and abC appeared to be sequestered atthe cell membrane (Fig. 3B–D). However, at day 4 of differ-entiation, abC expression appeared to increase within 25 and40 rpm EBs, and altered localization of abC was suggested bythe appearance of abC + nuclei, which was distinct from abCpreviously observed at the cell membrane (Fig. 3H, I). Ingeneral, 55 rpm EBs exhibited little dephosphorylated b-ca-tenin, and the majority of b-catenin expression was restrictedto the cell membrane at days 2 and 4 of differentiation (Fig.3D, J). After 7 days of differentiation, the expression of b-catenin was significantly decreased in all rotary conditionscompared with static EBs, and little to no nuclear staining ofb-catenin was observed in EBs from any of the culture con-ditions (Fig. 3M–P), suggesting that b-catenin transcriptionalactivity was diminished. Overall, patterns of b-catenin ex-pression within differentiating EBs indicated that ESC ag-

gregation modulates both the expression patterns andphosphorylation state of b-catenin.

Interestingly, a comparison of the dynamic expressionpatterns of E-cadherin (Fig. 2) and b-catenin (Fig. 3) overtime revealed significant correlations, including a positivecorrelation (P < 0.01) between E-cadherin and abC after 2days of differentiation and a negative correlation (P < 0.03)between E-cadherin and total b-catenin expression after4 days of differentiation (Table 1). Together, such resultssuggest an interplay between intercellular adhesions and b-catenin, with increased early expression of E-cadherin, fol-lowed by decreased E-cadherin levels, supporting increasedexpression of abC and total b-catenin, respectively.

b-catenin cellular localization in response to EBformation kinetics

The intracellular localization of total b-catenin was furtherexamined by western blot analysis of cytoplasmic (Fig. 4A)and nuclear (Fig. 4B) protein fractions from EBs culturedin static or rotary (25, 40, and 55 rpm) conditions at days 2and 4 of differentiation. EBs from all conditions expressedtotal b-catenin in both the cytoplasm and nucleus at all timepoints examined (Fig. 4A, B). Overall, the expression ofb-catenin in the cytoplasm remained relatively constantacross all conditions at both time points examined (Fig. 4A).In contrast, nuclear fractions suggested differences in the

FIG. 1. b-catenin and E-cadherin expression patternswithin embryonic stem cells(ESCs). Localization of (A)nuclei, (B) E-cadherin, and(C) b-catenin in undifferenti-ated ESCs demonstrates ex-pression primarily at the cellmembrane between adjacentcells (D). Green = E-cadherin,red =b-catenin, blue = nuclei.Scale bar = 50mm. Color ima-ges available online at www.liebertpub.com/scd


b-catenin expression across different EB formation conditions.Specifically, EBs maintained at 25 rpm appeared to exhibitincreased nuclear expression of total b-catenin at both days 2and 4 of differentiation compared with other rotary conditions(Fig. 4B), which was consistent with the increased expressionof abC noted by immunofluorescence in 25 rpm EBs at day 4 ofdifferentiation (Fig. 3F). Taken together, these data suggest thedynamic regulation of b-catenin protein expression, phos-phorylation state, and localization during the initial stages ofdifferentiation in response to intercellular adhesion kinetics.

TCF/LEF activity in response to ESCaggregation kinetics

To quantitatively assess b-catenin transcriptional activitywith high temporal resolution during the initial stages ofEB differentiation, ESCs were transduced with a TCF/LEF-luciferase lentivirus. Transduced cells retained a typical un-differentiated morphological appearance at all MOI levelsexamined (Supplementary Fig. S1A–C); however, only ESCstransduced with 10 and 25 MOI survived after puromycintreatment (Supplementary Fig. S1D–F). Stably transduced

clones were characterized based on morphological appear-ance, doubling time, pluripotency (alkaline phosphatase ac-tivity and Oct-4 protein expression), luciferase expression, andthe capacity to form EBs (Supplementary Fig. S2), all of whichsupported the maintenance of the pluripotent ESC phenotype.The clones that exhibited the greatest dynamic range in lu-ciferase activity in response to LiCl treatment were selected foradditional analyses (Supplementary Fig. S2G). Importantly,transduced ESCs formed and maintained EBs that were sim-ilar in size and appearance to EBs formed from naıve ESCs[36] at various rotary speeds (25, 40 and 55 rpm) and in staticconditions (Supplementary Fig. 2H), indicating the capacity toquantitatively assess b-catenin transcriptional activity in re-sponse to ESC aggregation kinetics.

EBs cultured under all conditions exhibited transientb-catenin transcriptional activity during the first 2–6 days ofdifferentiation (Fig. 4C). After 2 days of EB differentia-tion, static and 25 rpm EBs exhibited the highest levels ofluciferase activity, compared with 40 and 55 rpm (P < 0.05).By day 3, EBs from all rotary conditions exhibited increasedb-catenin transcriptional activity compared with static cul-tures (P < 0.001, 25 rpm; P < 0.01, 40 rpm; P < 0.05, 55 rpm),

FIG. 2. E-cadherin expression patterns during embryoid body (EB) differentiation. Static (A, F, K, P) and rotary EBs at 25 (B,G, L, Q), 40 (C, H, M, R), and 55 rpm (D, I, N, S) were stained for E-cadherin + cells at days 1 (A–D), 2 (F–I), 4 (K–N), and 7(P–S) of differentiation. Quantification of the staining patterns of E-cadherin (E, J, O, T), suggesting that E-cadherin ex-pression within EBs was dynamically modulated during differentiation as a result of changes in ESC aggregation kinetics.Scale bar = 50 mm. Green = E-cadherin, blue = nuclei. n = 3, *P < 0.05. Color images available online at www.liebertpub.com/scd


with the b-catenin transcription in 25 rpm EBs statisticallyincreased compared with the other two rotary conditions(P < 0.05). Although EBs cultured in rotary orbital suspensionincreased transcription activity between days 2 and 3 ofdifferentiation, statically cultured EBs exhibited decreasedTCF/LEF activity over the same time course (Fig. 4C).Within 6 days of differentiation, all cultures exhibited de-creased b-catenin transcription, below the initial levels at day2, though static EBs maintained an increased level of TCF/LEF activity compared with any of the rotary orbital cultureconditions (P < 0.001) (Fig. 4C). Overall, the TCF/LEF tran-scriptional activity was most active within the first 3 days of

EB differentiation and increasing the EB aggregation kineticsvia rotary orbital conditions (25 rpm) enhanced the peakb-catenin transcriptional activity between days 2 and 4 ofdifferentiation, consistent with immunofluorescence andwestern blot, which together reflected increased b-cateninprotein expression and nuclear localization.

Temporal dynamics of b-catenin-targetgene expression

Since b-catenin expression, phosphorylation state, locali-zation, and transcriptional activity were modulated by

FIG. 3. Expression and phosphorylation state of b-catenin within EBs. Static (A, G, M) and rotary EBs at 25 (B, H, N), 40 (C,I, O), and 55 rpm (D, J, P) were stained for b-catenin + and abC + cells at days 2 (A–D), 4 (G–J), and 7 (M–P) of differen-tiation. Quantification of the expression of b-catenin (E, K, Q) and abC (F, L, R) indicated differences in b-catenin phos-phorylation and location during differentiation. Scale bar = 20 mm. Red = b-catenin, green = abC, yellow = b-catenin/abCoverlay, blue = nuclei. n = 3, *P < 0.05. Color images available online at www.liebertpub.com/scd

FIG. 4. b-catenin cellular localization and T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) activity in differentiatingEBs. Although cytoplasmic (A) expression of b-catenin was similar among culture conditions, nuclear (B) b-catenin exhibitedchanges in expression among static and rotary (25, 40, 55 rpm) EBs after days 2 and 4 of differentiation. TCF/LEF-mediatedluciferase activity was transiently increased by rotary orbital culture compared with static EBs, with a peak in expression after 3days of differentiation (C); within rotary conditions, EBs maintained at 25 rpm yielded the largest increase in luciferase activity.P < 0.05, #compared with all other conditions, {compared with 40 rpm, and {{compared with 55 rpm. Color images available onlineat www.liebertpub.com/scd


ESC aggregation kinetics, the downstream expression ofb-catenin-target genes was examined. The expression pat-terns of canonical and noncanonical Wnt genes (Wnt3a andWnt11, respectively) exhibited similar temporal expression(Fig. 5A, B). After 2 days of differentiation, EBs from allculture conditions expressed similar levels of both Wnt11and Wnt3a; however, after 4 days of differentiation, 25 rpmEBs significantly decreased Wnt11 expression compared withstatic and 55 rpm (P < 0.05, static; P < 0.01, 55 rpm), accom-panied by a decrease in Wnt3a expression compared with55 rpm (P < 0.05). At day 6 of differentiation, 40 rpm EBsexhibited increased Wnt11 and Wnt3a expression levelscompared with all other conditions (P < 0.05) (Fig. 5A, B).Interestingly, expression of Dkk-1, a canonical Wnt inhibitor,increased in 25 rpm EBs at days 4 and 8 compared with staticand 55 rpm EBs (P < 0.05) (Fig. 5A–C). Moreover, Dkk-1 ex-pression increased in all rotary conditions compared withstatic culture at day 4 of differentiation (P < 0.001, 25 rpm;P < 0.01, 40 rpm; P < 0.05, 55 rpm) (Fig. 5C).

Expression of Brachyury-T (B-T), an early marker associatedwith primitive streak formation and mesoderm differentiation,was increased significantly after 2 days of differentiationwithin 25 rpm EBs compared with all other culture conditions

(P < 0.05), followed by increased expression at day 4 in allrotary conditions compared with static culture (P < 0.01). Me-soderm posterior 1 (Mesp-I), a direct target of TCF/LEF regu-lated transcription [40], which is also implicated in earlymesoderm differentiation within ESCs [10], exhibited in-creased expression in 25 rpm conditions at day 4 of differen-tiation compared with all other culture conditions (P < 0.001).

Temporally controlled Wnt/b-catenin signaling has beenimplicated in the specification of mesoderm cells towardcardiac progenitors and subsequent cardiomyocyte matura-tion [27,41,42]. Nkx2.5, an early marker of cardiomyocyteprogenitors, exhibited increased expression at early timepoints in all rotary orbital culture conditions compared withstatic cultures (Fig. 5G, H). After 6 days of differentiation,Nkx2.5 expression was greater in all rotary conditions com-pared with static culture (P < 0.005, 25 rpm; P < 0.001, 40 rpm;P < 0.05, 55 rpm), and Nkx2.5 continued to exhibit increasedexpression in 25 rpm EBs compared with all other cultureconditions after 10 days of differentiation (P < 0.005). Like-wise, 25 rpm EBs expressed increased levels of Myocyte en-hancer 2c (Mef-2c) after 10 days of differentiation comparedwith all other conditions (P < 0.005, static; P < 0.01, 40 &55 rpm).

FIG. 5. Expression of b-catenin-target cardiomyogenic genes. qPCR analysis of RNA from static and rotary EBs (25, 40, 55 rpm)was performed at day 2, 4, 6, 8, and 10 of differentiation. Expression levels of noncanonical and canonical Wnts (A, B) were similar,but were modulated by culture condition, with 25 rpm rotary conditions exhibiting decreased levels of both Wnt11 and Wnt3a. Incontrast, Wnt inhibitor Dkk-1 expression was increased within rotary EBs compared with static EBs (C), also corresponding toincreased mesoderm-related gene expression within rotary conditions compared with static (D, E). In addition, early cardio-myogenic markers Mef-2c and Nkx2.5 were increased by rotary orbital culture (F, G), with 25 rpm conditions resulting in increasedexpression of sarcomeric muscle genes a-MHC and MLC-2v (H, I). n = 3, P < 0.05, #compared with all other conditions, *comparedwith static, **compared with 25 rpm, and {{compared with 55 rpm. Color images available online at www.liebertpub.com/scd


Expression of sarcomeric muscle-related genes a-myosinheavy chain (a-MHC) and myosin light chain 2 ventricle (MLC-2v), phenotypic markers of more mature cardiomyocytes,were observed by day 10 of differentiation within EBsmaintained at 25 rpm (Fig. 5H, I). MLC-2v expression wassignificantly increased within 25 rpm EBs compared withother culture conditions (P < 0.05, static; P < 0.01, 40 &55 rpm) as early as day 8 of differentiation compared withother culture conditions, and continued to exhibit increasedexpression after 10 days of differentiation (P < 0.005) (Fig. 5I).Together, the targets of the Wnt/b-catenin pathway (Mesp-1,Mef-2c) exhibited increased expression at early stages ofdifferentiation (day 4), followed by increased expression ofWnt and b-catenin inhibitors (Dkk-1, Nkx2.5) and cardio-myogenic gene transcription in response to accelerated ag-gregation kinetics within 25 rpm rotary orbital cultures.

Regulation of cardiomyogenic gene transcriptionby Wnt/b-catenin signaling

To establish a more mechanistic link between the observedregulation of b-catenin signaling and cardiomyogenic genetranscription, 25 rpm EBs were supplemented with inhibitorsof canonical Wnt/b-catenin signaling (IWP-4; 5 mM) duringthe initial 4 days of differentiation (Fig. 6A). As expected, thepluripotency marker Oct-4 was significantly decreasedcompared with ESCs, and expression of genes related toendoderm (Foxa2) and ectoderm (Nestin) lineages was sig-nificantly increased compared with ESCs; however, Wnt in-hibition did not significantly affect the expression of genesrelated to pluripotency, endoderm, or ectoderm differentia-tion. In contrast, inhibition of Wnt signaling significantlyabrogated the previously observed increase in cardiomyo-genic genes a-MHC and MLC-2v, thus indicating the role ofcanonical Wnt/b-catenin signaling in mediating the cardio-

myogenic gene transcription downstream of intercellularaggregation at 25 rpm. The results of this study suggest thatthe dynamic interplay between E-cadherin-mediated cellularadhesion and Wnt signaling leads to nuclear accumula-tion and TCF/LEF transcriptional activation via depho-sphorylated, b-catenin (abC), thereby inducing downstreamcardiomyogenic gene expression (Fig. 6B).

Discussion

In this study, b-catenin expression, localization, andtranscriptional activity, as well as cardiomyogenic genetranscription were assessed in response to changes in ESCaggregation kinetics. Overall, rotary orbital culture increasedb-catenin-regulated transcription and cardiomyogenic geneexpression compared with static culture conditions. Thespatiotemporal location and phosphorylation state of b-catenin was modulated by rotary orbital speed, with slowerrotary speeds (faster ESC aggregation kinetics), resultingin increased accumulation of nuclear, dephosphorylated b-catenin at early time points (Figs. 3 and 4). In addition, genetranscription downstream of b-catenin was increased initiallyby slower rotary speeds, evidenced by increased TCF/LEFactivity and the expression of Mesp-1 and Mef-2c (Fig. 5).Moreover, inhibition of Wnt signaling significantly de-creased cardiomyogenic gene transcription. Overall, thisstudy illustrates that b-catenin signaling and downstreamcardiomyogenic gene transcription are enhanced withinculture conditions that promote increased multicellular ag-gregation kinetics.

Recent studies have illustrated that activation of the b-catenin pathway can play both inductive and repressive rolesin cardiomyogenesis, depending on the temporal onset andduration of transcriptional activity [28,42,43]. Early activa-tion of b-catenin within differentiating ESCs has been linked

FIG. 6. Influence of canonical Wnt/b-catenin signaling in aggregation-mediated cardiogenic gene transcription. Expression ofgenes related to pluripotency (Oct-4), endoderm (Foxa2), and ectoderm (Nestin) phenotypes was not altered by inhibition of Wntsignaling via treatment with IWP-4 during the first 4 days of differentiation at 25 rpm; however, cardiomyogenic markers a-MHC and MLC-2v were significantly decreased on Wnt inhibition (A). The proposed model depicting Wnt/b-catenin-mediatedincrease in cardiomyogenic gene transcription resulting from intercellular adhesion of ESCs (B). n = 3, *P < 0.05 *compared tomatched treatment and #compared to undifferentiated ESCs.


to increased mesoderm differentiation and proliferation ofMesp-1 + , Isl-1 + , and Mef-2c + cardiomyocyte progenitorcells [10,30,42–45]. However, persistent b-catenin signaling(i.e., beyond day 4 of differentiation) inhibits subsequentcardiomyocyte maturation [42,43]; conversely, inhibition ofb-catenin signaling through natural (e.g., Dkk-1) or small-molecule Wnt inhibitors at later stages of differentiation (day4 + ) increases mature cardiomyocyte development withinEBs [28,46–48]. A similar biphasic regulation of b-cateninactivity and inhibitor expression was observed in this studyin response to accelerated cellular adhesion kinetics. Nucleartotal b-catenin expression was modulated by ESC aggrega-tion kinetics (Fig. 4B), with increased expression of signalingactive b-catenin within 25 rpm rotary conditions at early timepoints of differentiation (days 2 and 4, Fig. 3), significantlyincreased TCF/LEF activation at day 3 of differentiation (Fig.4C), and increased Mesp-1 and Mef-2c expression (day 4)compared with other conditions (Fig. 5). Subsequently, ex-pression of b-catenin signaling inhibitors Dkk-1 and Nkx2.5[49] was also increased within the same rotary orbital con-dition, concomitant with decreased Wnt expression levels(days 4–6). The increase in Dkk-1 expression may be relatedto increased Mesp-1 expression within 25 rpm rotary condi-tions, as Mesp-1 is a transcription factor that is capableof directly increasing Dkk-1 expression [40]. Thus, the ex-pression of Mesp-1, Dkk-1, and Nkx2.5 at later stages of dif-ferentiation supports the maturation of cardiomyocyteprogenitors, as evidenced by increased expression of MLC-2vin 25 rpm rotary conditions (Fig. 5I). Consistent with ourprevious results [36,37], rotary orbital culture promotedcardiomyogenic gene transcription in a speed-dependentmanner, and the results from this study suggest that b-catenin signaling may be implicated in the regulation ofmesoderm differentiation and maturation of cardiomyocyteprogenitors (Fig. 6) in response to changes in early ESC ag-gregation kinetics.

Until recently, the membrane-associated and nuclear sig-naling roles of b-catenin have been largely studied inde-pendently; however, increasing evidence suggests that thetwo processes may be intimately coordinated through com-petition for the total pool of available b-catenin in the cell[31,33]. Studies using recombinant forms of b-catenin indi-cate that the binding domains of b-catenin which mediateinteractions with cadherins, TCF/LEF, and APC are mutu-ally exclusive [50]. The loss of E-cadherin expression is cor-related with invasive phenotypes during cancer progressionand is exhibited concomitant with up-regulation of the Wnt/b-catenin signaling, suggesting the possible interplay be-tween E-cadherin expression and b-catenin transcriptionalactivation [12,51–55]. In the undifferentiated state, ESCslargely express b-catenin at the plasma membrane, presum-ably bound to E-cadherin, with little nuclear localization andsignaling (Fig. 1) [39]. Moreover, the culture of ESCs in athree-dimensional microwell format increased E-cadherinexpression and subsequently increased Wnt signaling on EBformation, suggesting a link between intercellular adhesions,Wnt signaling, and cardiogenesis in ESCs [56]. Similarly, inthis study, E-cadherin was dynamically regulated, includingsignificant correlations between E-cadherin (Fig. 2) and b-catenin (Fig. 3) expression over time (Table 1), concurrentwith increased TCF/LEF transcriptional activity (Fig. 4C).The regulation of intercellular adhesions in EBs is consistent

with changes during embryonic development, whereby E-cadherin is down-regulated as cells traverse the primitivestreak and b-catenin signaling is increased to promote axisformation and mesoderm differentiation [57–62]. It is likelythat similar interrelated signaling between cadherins and b-catenin may enable the pool of cadherin-bound b-catenin tobecome available for nuclear translocation and signaling onremodeling of cadherins after EB formation. Although thedirect relationship between cadherin and TCF/LEF-medi-ated regulation of the b-catenin pool remains unclear[33,63,64], evidence from this study suggests a correlationbetween E-cadherin and b-catenin signaling during EB for-mation and ESC differentiation.

Rotary orbital suspension culture has previously demon-strated the modulation of EB formation kinetics across arange of mixing conditions, with slower speeds resulting inincreased EB formation kinetics [34,36]. The changes in b-catenin transcriptional activity as a function of rotary speed,in conjunction with changes in E-cadherin expression, indi-cate that cell association kinetics during EB formation canimpact the subsequent pathway activation and ESC differ-entiation. b-catenin transcriptional activity was significantlyincreased at early stages of differentiation in EBs that exhibitfaster cell association kinetics during EB formation at slowerrotary speeds. Studies indicate that mixed culture conditionsalso modulate EB size, which may be partially responsiblefor changes in ESC differentiation [36]. Mesoderm inductionin different sized EBs, however, is thought to result fromparacrine actions of endoderm cells at the exterior of EBs[65], which do not arise until later stages of differentiation. Inthe context of this study, the initial cell specification resultingfrom changes in b-catenin signaling at early time points(days 2–4) is likely independent of signaling from divergentcell populations. The observed changes in cell aggregationkinetics due to rotary orbital suspension culture conditionsmay also be modulated across a range of suspension cultureformats [66]; b-catenin transcriptional activity may be im-plicated in the modulation of cardiogenic differentiation as afunction of bioreactor configuration, mixing speed, and celldensity [2,67]. Hanging drop cultures exhibit an increasedendogenous propensity to differentiate toward mesodermlineages and develop functional cardiomyocytes comparedwith other formats [37,68]. In contrast to suspension cultures,which rely on the random collision of cells in larger-volumesuspensions to form EBs, hanging-drop cultures force ESCaggregation in small volumes to more precisely controlEB size, which likely results in faster association kineticscompared with suspension cultures. The increased cardiacdifferentiation yielded by hanging drops, therefore, is con-sistent with the observed increase in cardiomyogenic genetranscription in response to slower rotary speed and fasteraggregation kinetics. Many recently developed technologiesfor spheroid formation may also alter the formation kinetics,by controlling aggregation within microwells or on micro-patterned surfaces [69–71]; EBs formed via forced aggre-gation exhibited increased homogeneity and endogenousdifferentiation capacities across a similar range of rotaryconditions examined in this study [35]. Thus, techniques tocontrol intercellular adhesion dynamics may impact thedifferentiation capacity of stem and progenitor cells to eitheraid or hinder directed differentiation protocols, dependingon the application. The results from this study, coupled with


the developing understanding of the complex relationshipbetween E-cadherin and b-catenin, highlight the importantroles of b-catenin signaling in ESCs, which may be modu-lated by increasing cell association kinetics during EB for-mation to enhance cardiomyogenic differentiation.

Acknowledgments

This work was supported by the National Institutes ofHealth (NIH R01 EB010061) and by the National ScienceFoundation (NSF CBET 0939511). M.A.K. is currently sup-ported by an American Heart Association (AHA) Pre-Doc-toral Fellowship and earlier by an NSF Graduate ResearchFellowship. C.Y.S. was also previously supported by an NSFGraduate Research Fellowship and an AHA Pre-DoctoralFellowship.

Author Disclosure Statement

No competing financial interests exist.

References

1. Larue L, C Antos, S Butz, O Huber, V Delmas, M Dominisand R Kemler. (1996). A role for cadherins in tissue forma-tion. Development 122:3185–3194.

2. Itskovitz-Eldor J, M Schuldiner, D Karsenti, A Eden, O Yanuka,M Amit, H Soreq and N Benvenisty. (2000). Differentiation ofhuman embryonic stem cells into embryoid bodies compro-mising the three embryonic germ layers. Mol Med 6:88–95.

3. Doetschman TC, H Eistetter, M Katz, W Schmidt and RKemler. (1985). The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolksac, blood islands and myocardium. J Embryol Exp Morphol87:27–45.

4. Keller G. (2005). Embryonic stem cell differentiation: emer-gence of a new era in biology and medicine. Genes Dev19:1129–1155.

5. Yost C, M Torres, JR Miller, E Huang, D Kimelman and RTMoon. (1996). The axis-inducing activity, stability, and subcel-lular distribution of beta-catenin is regulated in Xenopus em-bryos by glycogen synthase kinase 3. Genes Dev 10:1443–1454.

6. Mohamed OA, HJ Clarke and D Dufort. (2004). Beta-cateninsignaling marks the prospective site of primitive streak for-mation in the mouse embryo. Dev Dyn 231:416–424.

7. Haegel H, L Larue, M Ohsugi, L Fedorov, K Herrenknechtand R Kemler. (1995). Lack of beta-catenin affects mousedevelopment at gastrulation. Development 121:3529–3537.

8. Sato N, L Meijer, L Skaltsounis, P Greengard and AH Bri-vanlou. (2004). Maintenance of pluripotency in human andmouse embryonic stem cells through activation of Wnt sig-naling by a pharmacological GSK-3-specific inhibitor. NatMed 10:55–63.

9. Kelly KF, DY Ng, G Jayakumaran, GA Wood, H Koide andBW Doble. (2011). B-catenin enhances Oct-4 activity andreinforces pluripotency through a TCF-independent mech-anism. Cell Stem Cell 8:214–227.

10. Lindsley RC, JG Gill, M Kyba, TL Murphy and KM Murphy.(2006). Canonical Wnt signaling is required for developmentof embryonic stem cell-derived mesoderm. Development133:3787–3796.

11. ten Berge D, W Koole, C Fuerer, M Fish, E Eroglu and RNusse. (2008). Wnt signaling mediates self-organization andaxis formation in embryoid bodies. Cell Stem Cell 3:508–518.

12. Gottardi CJ and BM Gumbiner. (2004). Distinct molecularforms of beta-catenin are targeted to adhesive or transcrip-tional complexes. J Cell Biol 167:339–349.

13. Kemler R. (1993). From cadherins to catenins: cytoplasmicprotein interactions and regulation of cell adhesion. TrendsGenet 9:317–321.

14. Aberle H, H Schwartz and R Kemler. (1996). Cadherin-ca-tenin complex: protein interactions and their implications forcadherin function. J Cell Biochem 61:514–523.

15. Yamada S, S Pokutta, F Drees, WI Weis and WJ Nelson.(2005). Deconstructing the cadherin-catenin-actin complex.Cell 123:889–901.

16. Henderson BR. (2000). Nuclear-cytoplasmic shuttling ofAPC regulates beta-catenin subcellular localization andturnover. Nat Cell Biol 2:653–660.

17. Behrens J, JP von Kries, M Kuhl, L Bruhn, D Wedlich, RGrosschedl and W Birchmeier. (1996). Functional interactionof beta-catenin with the transcription factor LEF-1. Nature382:638–642.

18. Clevers H. (2006). Wnt/beta-catenin signaling in develop-ment and disease. Cell 127:469–480.

19. van Noort M, J Meeldijk, R van der Zee, O Destree and HClevers. (2002). Wnt signaling controls the phosphorylationstatus of beta-catenin. J Biol Chem 277:17901–17905.

20. Hart MJ, R de los Santos, IN Albert, B Rubinfeld and PPolakis. (1998). Downregulation of beta-catenin by humanAxin and its association with the APC tumor suppressor,beta-catenin and GSK3 beta. Curr Biol 8:573–581.

21. Orford K, C Crockett, JP Jensen, AM Weissman and SWByers. (1997). Serine phosphorylation-regulated ubiquitina-tion and degradation of beta-catenin. J Biol Chem 272:24735–24738.

22. Cong F, L Schweizer and H Varmus. (2004). Wnt signalsacross the plasma membrane to activate the beta-cateninpathway by forming oligomers containing its receptors,frizzled and LRP. Development 131:5103–5115.

23. Mao J, J Wang, B Liu, W Pan, GH Farr, C Flynn, H Yuan, STakada, D Kimelman, L Li and D Wu. (2001). Low-densitylipoprotein receptor-related protein-5 binds to Axin andregulates the canonical Wnt signaling pathway. Mol Cell7:801–809.

24. Munemitsu S, I Albert, B Souza, B Rubinfeld and P Polakis.(1995). Regulation of intracellular beta-catenin levels by theadenomatous polyposis coli (APC) tumor-suppressor pro-tein. Proc Natl Acad Sci (USA) 92:3046–3050.

25. Korinek V, N Barker, PJ Morin, D van Wichen, R de Weger,KW Kinzler, B Vogelstein and H Clevers. (1997). Con-stitutive transcriptional activation by a beta-catenin-tcfcomplex in APC-/- colon carcinoma. Science 275:1784–1787.

26. Hurlstone AFL, A-PG Haramis, E Wienholds, H Begthel, JKorving, F Van Eeden, E Cuppen, D Zivkovic, RHA Plasterkand H Clevers. (2003). The Wnt/beta-catenin pathway reg-ulates cardiac valve formation. Nature 425:633–637.

27. Lin L, L Cui, W Zhou, D Dufort, X Zhang, C-L Cai, L Bu, LYang, J Martin, et al. (2007). Beta-catenin directly regulatesIslet1 expression in cardiovascular progenitors and is re-quired for multiple aspects of cardiogenesis. Proc Natl AcadSci (USA) 104:9313–9318.

28. Lian X, C Hsiao, G Wilson, K Zhu, LB Hazeltine, SM Azarin,KK Raval, J Zhang, TJ Kamp and SP Palecek. (2012). Robustcardiomyocyte differentiation from human pluripotent stemcells via temporal modulation of canonical Wnt signaling.Proc Natl Acad Sci (USA) 109:E1848–E1857.


29. Nakamura T, M Sano, Z Songyang and MD Schneider.(2003). A Wnt- and beta -catenin-dependent pathway formammalian cardiac myogenesis. Proc Natl Acad Sci (USA)100:5834–5839.

30. Paige SL, T Osugi, OK Afanasiev, L Pabon, H Reinecke andCE Murry. (2010). Endogenous Wnt/beta-catenin signalingis required for cardiac differentiation in human embryonicstem cells. PLoS One 5:e11134.

31. Nelson WJ and R Nusse. (2004). Convergence of Wnt, beta-catenin, and cadherin pathways. Science 303:1483–1487.

32. Maretzky T, K Reiss, A Ludwig, J Buchholz, F Scholz, EProksch, B de Strooper, D Hartmann and P Saftig. (2005).ADAM10 mediates E-cadherin shedding and regulates epi-thelial cell-cell adhesion, migration, and beta-catenin trans-location. Proc Natl Acad Sci (USA) 102:9182–9187.

33. Kam Y and V Quaranta. (2009). Cadherin-bound beta-catenin feeds into the Wnt pathway upon adherens junctionsdissociation: evidence for an intersection between beta-catenin pools. PLoS ONE 4:e4580.

34. Carpenedo RL, CY Sargent and TC McDevitt. (2007). Rotarysuspension culture enhances the efficiency, yield, and ho-mogeneity of embryoid body differentiation. Stem Cells25:2224–2234.

35. Kinney MA, R Saeed and TC McDevitt. (2012). Systematicanalysis of embryonic stem cell differentiation in hydrody-namic environments with controlled embryoid body size. IntBiol 4:641–650.

36. Sargent CY, GY Berguig, MA Kinney, LA Hiatt, RL Carpe-nedo, RE Berson and TC McDevitt. (2010). Hydrodynamicmodulation of embryonic stem cell differentiation by rotaryorbital suspension culture. Biotechnol Bioeng 105:611–626.

37. Sargent CY, GY Berguig and TC McDevitt. (2009). Cardio-myogenic differentiation of embryoid bodies is promoted byrotary orbital suspension culture. Tissue Eng Part A 15:331–342.

38. Carpenter AE, TR Jones, MR Lamprecht, C Clarke, IH Kang,O Friman, DA Guertin, JH Chang, RA Lindquist, et al.(2006). CellProfiler: image analysis software for identifyingand quantifying cell phenotypes. Genome Biol 7:R100.

39. Anton R, HA Kestler and M Kuhl. (2007). Beta-catenin signal-ing contributes to stemness and regulates early differentiationin murine embryonic stem cells. FEBS Lett 581:5247–5254.

40. David R, C Brenner, J Stieber, F Schwarz, S Brunner, MVollmer, E Mentele, J Muller-Hocker, S Kitajima, et al.(2008). MesP1 drives vertebrate cardiovascular differentia-tion through Dkk-1-mediated blockade of Wnt-signalling.Nat Cell Biol 10:338–345.

41. Kwon C, J Arnold, EC Hsiao, MM Taketo, BR Conklin and DSrivastava. (2007). Canonical Wnt signaling is a positiveregulator of mammalian cardiac progenitors. Proc Natl AcadSci (USA) 104:10894–10899.

42. Naito AT, I Shiojima, H Akazawa, K Hidaka, T Morisaki, AKikuchi and I Komuro. (2006). Developmental stage-specificbiphasic roles of Wnt/beta-catenin signaling in cardiomyo-genesis and hematopoiesis. Proc Natl Acad Sci (USA) 103:19812–19817.

43. Ueno S, G Weidinger, T Osugi, AD Kohn, JL Golob, L Pabon,H Reinecke, RT Moon and CE Murry. (2007). Biphasic rolefor Wnt/beta-catenin signaling in cardiac specification inzebrafish and embryonic stem cells. Proc Natl Acad Sci(USA) 104:9685–9690.

44. Kwon C, L Qian, P Cheng, V Nigam, J Arnold and D Sri-vastava. (2009). A regulatory pathway involving Notch1/beta-catenin/Isl1 determines cardiac progenitor cell fate.Nat Cell Biol 11:951–957.

45. Qyang Y, S Martin-Puig, M Chiravuri, S Chen, H Xu, L Bu,X Jiang, L Lin, A Granger, et al. (2007). The renewal anddifferentiation of Isl1 + cardiovascular progenitors are con-trolled by a Wnt/beta-catenin pathway. Cell Stem Cell1:165–179.

46. Ren Y, MY Lee, S Schliffke, J Paavola, PJ Amos, X Ge, M Ye,S Zhu, G Senyei, et al. (2011). Small molecule Wnt inhibitorsenhance the efficiency of BMP-4-directed cardiac differenti-ation of human pluripotent stem cells. J Mol Cell Cardiol51:280–287.

47. Wang H, J Hao and CC Hong. (2011). Cardiac induction ofembryonic stem cells by a small molecule inhibitor of Wnt/b-catenin signaling. ACS Chem Biol 6:192–197.

48. Willems E, S Spiering, H Davidovics, M Lanier, Z Xia, MDawson, J Cashman and M Mercola. (2011). Small-moleculeinhibitors of the Wnt pathway potently promote cardio-myocytes from human embryonic stem cell-derived meso-derm. Circ Res 109:360–364.

49. Riazi AM, JK Takeuchi, LK Hornberger, SH Zaidi, F Amini, JColes, BG Bruneau and GS Van Arsdell. (2009). NKX2-5regulates the expression of beta-catenin and GATA4 inventricular myocytes. PLoS One 4:e5698.

50. Orsulic S, O Huber, H Aberle, S Arnold and R Kemler.(1999). E-cadherin binding prevents beta-catenin nuclearlocalization and beta-catenin/LEF-1-mediated transactiva-tion. J Cell Sci 112:1237–1245.

51. Gavard J and R-M Mege. (2005). Once upon a time therewas beta-catenin in cadherin-mediated signalling. Biol Cell97:921–926.

52. Palmer HG, JM Gonzalez-Sancho, J Espada, MT Berciano, IPuig, J Baulida, M Quintanilla, A Cano, AG de Herreros, MLafarga and A Munoz. (2001). Vitamin D(3) promotes thedifferentiation of colon carcinoma cells by the induction of E-cadherin and the inhibition of beta-catenin signaling. J CellBiol 154:369–387.

53. Jamora C and E Fuchs. (2002). Intercellular adhesion, sig-nalling and the cytoskeleton. Nat Cell Biol 4:E101-8.

54. Nakanishi Y, A Ochiai, S Akimoto, H Kato, H Watanabe, YTachimori, S Yamamoto and S Hirohashi. (1997). Expressionof E-cadherin, alpha-catenin, beta-catenin and plakoglobinin esophageal carcinomas and its prognostic significance:immunohistochemical analysis of 96 lesions. Oncology 54:158–165.

55. Gottardi CJ, E Wong and BM Gumbiner. (2001). E-cadherinsuppresses cellular transformation by inhibiting beta-cateninsignaling in an adhesion-independent manner. J Cell Biol153:1049–1060.

56. Azarin SM, X Lian, EA Larson, HM Popelka, JJ De Pablo andSP Palecek. (2012). Modulation of Wnt/b-catenin signalingin human embryonic stem cells using a 3-D microwell array.Biomaterials 33:2041–2049.

57. Burdsal CA, CH Damsky and RA Pedersen. (1993). The roleof E-cadherin and integrins in mesoderm differentiation andmigration at the mammalian primitive streak. Development118:829–844.

58. Cadigan KM and R Nusse. (1997). Wnt signaling: a commontheme in animal development. Genes Dev 11:3286–3305.

59. Ciruna B and J Rossant. (2001). FGF signaling regulatesmesoderm cell fate specification and morphogenetic move-ment at the primitive streak. Dev Cell 1:37–49.

60. Hecht A, CM Litterst, O Huber and R Kemler. (1999).Functional characterization of multiple transactivatingelements in beta-catenin, some of which interact with theTATA-binding protein in vitro. J Biol Chem 274:18017–18025.


61. Hecht A, K Vleminckx, MP Stemmler, F van Roy and RKemler. (2000). The p300/CBP acetyltransferases function astranscriptional coactivators of beta-catenin in vertebrates.EMBO J 19:1839–1850.

62. Barker N, A Hurlstone, H Musisi, A Miles, M Bienz and HClevers. (2001). The chromatin remodelling factor Brg-1 in-teracts with beta-catenin to promote target gene activation.EMBO J 20:4935–4943.

63. Bonvini P, WG An, A Rosolen, P Nguyen, J Trepel, A Garcia deHerreros, M Dunach and LM Neckers. (2001). Geldanamycinabrogates ErbB2 association with proteasome-resistant beta-catenin in melanoma cells, increases beta-catenin-E-cadherinassociation, and decreases beta-catenin-sensitive transcrip-tion. Cancer Res 61:1671–1677.

64. Marambaud P, J Shioi, G Serban, A Georgakopoulos, SSarner, V Nagy, L Baki, P Wen, S Efthimiopoulos, et al.(2002). A presenilin-1/gamma-secretase cleavage releasesthe E-cadherin intracellular domain and regulates disas-sembly of adherens junctions. EMBO J 21:1948–1956.

65. Bauwens CL, H Song, N Thavandiran, M Ungrin, S Masse, KNanthakumar, C Seguin and PW Zandstra. (2011). Geo-metric control of cardiomyogenic induction in human plu-ripotent stem cells. Tissue Eng Part A 17:1901–1909.

66. Kinney MA, CY Sargent and TC McDevitt. (2011). Themultiparametric effects of hydrodynamic environments onstem cell culture. Tissue Eng Part B Rev 17:249–262.

67. Shafa M, R Krawetz, Y Zhang, JB Rattner, A Godollei, HJDuff and DE Rancourt. (2011). Impact of stirred suspensionbioreactor culture on the differentiation of murine embry-onic stem cells into cardiomyocytes. BMC Cell Biol 12:53.

68. Yoon BS, SJ Yoo, JE Lee, S You, HT Lee and HS Yoon. (2006).Enhanced differentiation of human embryonic stem cellsinto cardiomyocytes by combining hanging drop cultureand 5-azacytidine treatment. Differentiation 74:149–159.

69. Mohr JC, JJ De Pablo and SP Palecek. (2006). 3-D microwellculture of human embryonic stem cells. Biomaterials 27:6032–6042.

70. Ungrin MD, C Joshi, A Nica, C Bauwens and PW Zandstra.(2008). Reproducible, ultra high-throughput formation ofmulticellular organization from single cell suspension-derived human embryonic stem cell aggregates. PLoS One3:e1565.

71. Park J, CH Cho, N Parashurama, Y Li, F Berthiaume, MToner, AW Tilles and ML Yarmush. (2007). Microfabrication-based modulation of embryonic stem cell differentiation. LabChip 7:1018–1028.

Address correspondence to:Dr. Todd C. McDevitt

The Wallace H. Coulter Department of Biomedical EngineeringGeorgia Institute of Technology/Emory University

313 Ferst Drive, Suite 2102Atlanta, GA 30332-0532

E-mail: [email protected]

Received for publication January 4, 2013Accepted after revision May 9, 2013

Prepublished on Liebert Instant Online May 13, 2013


temporal modulation of β-catenin signaling by multicellular aggregation kinetics impacts embryonic...

Documents